Absolute side-chain structure at position 13 is required for the inhibitory activity of bromein.
Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu13 in the light chain was revealed to be essential for inhibition. Pro12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu13 are absolutely crucial for bromelain inhibition.
Sawano
Yoriko
Y
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Hatano
Ken-ichi
K
Miyakawa
Takuya
T
Tanokura
Masaru
M
D000595:Amino Acid Sequence; D031230:Ananas / Q000378:metabolism; D001963:Bromelains / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry*; D017353:Gene Deletion; D007930:Leucine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011392:Proline / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000737:chemistry
D015853:Cysteine Proteinase Inhibitors; D013912:Threonine; D001963:Bromelains; D011392:Proline; D003546:Cysteine Endopeptidases; C091760:stem bromelain; D007930:Leucine